The present invention relates to thermostable DNA polymerases derived from the thermophilic eubacterial species Thermoactinomyces vulgaris, as well as means for isolating and producing these enzymes. The thermostable DNA polymerases of the present invention are useful in many recombinant DNA techniques, including thermal cycle sequencing, nucleic acid amplification and reverse transcription.
Thermophilic bacteria are organisms which are capable of growth at elevated temperatures. Unlike the mesophiles, which grow best at temperatures in the range of 25-40xc2x0 C., or psychrophiles, which grow best at temperatures in the range of 15-20xc2x0 C., thermophiles grow best at temperatures greater than 50xc2x0 C. Indeed, some thermophiles grow best at 65-75xc2x0 C., and some of the hyperthermophiles grow at temperatures up to 130xc2x0 C. (e.g., J. G. Black, Microbiology Principles and Applications, 2d edition, Prentice Hall, N.J., 1993, p. 145-146).
The thermophilic bacteria encompass a wide variety of genera and species. There are thermophilic representatives included within the phototrophic bacteria (i.e., the purple bacteria, green bacteria, and cyanobacteria), eubacteria (i.e., Bacillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, lactic acid bacteria, actinomycetes, spirochetes, and numerous other genera), and the archaebacteria (i.e., Pyrococcus, Thermococcus, Thermoplasma, Thermotoga, Sulfolobus, and the methanogens). There are aerobic, as well as anaerobic thermophilic organisms. Thus, the environments in which thermophiles may be isolated vary greatly, although all of these organisms are isolated from areas associated with high temperatures. Natural geothermal habitats have a worldwide distribution and are primarily associated with tectonically active zones where major movements of the earth""s crust occur. Thermophilic bacteria have been isolated from all of the various geothermal habitats, including boiling springs with neutral pH ranges, sulfur-rich acidic springs, and deep-sea vents. In general, the organisms are optimally adapted to the temperatures at which they are living in these geothemal habitats (T. D. Brock, xe2x80x9cIntroduction: An overview of the thermophiles,xe2x80x9d in T. D. Brock (ed.), Thermophiles: General, Molecular and Applied Microbiology, John Wiley and Sons, New York, 1986, pp. 1-16). Basic, as well as applied research on thermophiles has provided some insight into the physiology of these organisms, as well as use of these organisms in industry and biotechnology.
Advances in molecular biology and industrial processes have led to increased interest in thermophilic organisms. Of particular interest has been the development of thermophilic enzymes for use in industries such as the detergent, flavor-enhancing, and starch industries. Indeed, the cost savings associated with longer storage stability and higher activity at higher temperatures of thermophilic enzymes, as compared to mesophilic enzymes, provide good reason to select and develop thermophilic enzymes for industrial and biotechnology applications. Thus, there has been much research conducted to characterize enzymes from thermophilic organisms. However, some thermophilic enzymes have less activity than their mesophilic counterparts under similar conditions at the elevated temperatures used in industry (typically temperatures in the range of 50-100xc2x0 C.) (T. K. Ng and William R. Kenealy, xe2x80x9cIndustrial Applications of Thermostable Enzymes,xe2x80x9d in T. D. Brock (ed.), Thermophiles: General, Molecular, and Applied Microbiology, 1986, John Wiley and Sons, New York, pp. 197-215). Thus, the choice of a thermostable enzyme over a mesophilic one may not be as beneficial as originally assumed. However, much research remains to be done to characterize and compare thermophilic enzymes of importance (e.g., polymerases, ligases, kinases, topoisomerases, restriction endonucleases, etc.) in areas such as molecular biology .
Extensive research has been conducted on isolation of DNA polymerases from mesophilic organisms such as E. coli. (e.g., Bessman et al., J. Biol. Chem. 223:171,1957; Buttin and Kornberg, J. Biol. Chem. 241:5419, 1966; and Joyce and Steitz, Trends Biochem. Sci., 12:288-292, 1987). Other mesophilic polymerases have also been studied, such as those of Bacillus licheniformis (Stenesh and McGowan, Biochim. Biophys. Acta 475:32-44, 1977; Stenesh and Roe, Biochim. Biophys. Acta 272:156-166, 1972); Bacillus subtilis (Low et al., J. Biol. Chem., 251:1311, 1976; and Ott et al., J. Bacteriol., 165:951, 1986); Salmonella typhimurium (Harwood et al., J. Biol. Chem., 245:5614, 1970; Hamilton and Grossman, Biochem., 13:1885, 1974); Streptococcus pneumoniae (Lopez et al., J. Biol. Chem., 264:4255, 1989); and Micrococcus luteus (Engler and Bessman, Cold Spring Harbor Symp., 43:929, 1979), to name but a few.
Somewhat less investigation has been performed on the isolation and purification of DNA polymerases from thermophilic organisms. However, native (i.e., non-recombinant) and/or recombinant thermostable DNA polymerases have been purified from various organisms, as shown in Table 1 below.
In addition to native forms, modified forms of thermostable DNA polymerases having reduced or absent 5xe2x80x2 to 3xe2x80x2 exonuclease activity have been expressed and purified from T. aquaticus, T. maritima. Thermus species sps17, Thermus species Z05, T. thermophilus, Bacillus stearothermophilus (U.S. Pat. Nos. 5,747,298, 5,834,253, 5,874,282, and 5,830,714) and T. africanus (WO 92/06200).
One application for thermostable DNA polymerases is the polymerase chain reaction (PCR). The PCR process is described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures of which are incorporated herein by reference. Primers, template, nucleoside triphosphates, appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves multiple cycles of denaturation of target DNA, hybridization of primers to the target DNA and synthesis of complementary strands. The extension product of each primer becomes a template in the subsequent cycle for production of the desired nucleic acid sequence. Use of a thermostable DNA polymerase enzyme in PCR allows repetitive heating/cooling cycles without the requirement of fresh DNA polymerase enzyme at each cooling step because heat will not destroy the polymerase activity. This represents a major advantage over the use of mesophilic DNA polymerase enzymes such as Klenow in PCR, as fresh mesophilic polymerase must be added to each individual reaction tube at every cycle. The use of Taq in PCR is described in U.S. Pat. No. 4,965,188, EP Publ. No. 258,017, and PCT Publ. No. 89/06691, herein incorporated by reference.
In addition to PCR, thermostable DNA polymerases are widely used in other molecular biology techniques including recombinant DNA methods. For example, various forms of Taq have been used in a combination method which utilizes reverse transcription and PCR (e.g., U.S. Pat. No. 5,322,770, herein incorporated by reference). DNA sequencing methods utilizing Taq DNA polymerase have also been described (e.g., U.S. Pat. No. 5,075,216, herein incorporated by reference).
However, some thermostable DNA polymerases have certain characteristics (e.g., 5xe2x80x2 to 3xe2x80x2 exonuclease activity) which are undesirable in PCR and other applications. In some cases, when thermostable DNA polymerases that have 5xe2x80x2 to 3xe2x80x2 exonuclease activity (e.g., Taq, Tma, Tsps17, TZ05, Tth and Taf) are used in the PCR process and other methods, a variety of undesirable results have been observed, including a limitation of the amount of PCR product produced, an impaired ability to generate long PCR products or to amplify regions containing significant secondary structure, the production of shadow bands or the attenuation in signal strength of desired termination bands during DNA sequencing, the degradation of the 5xe2x80x2 end of oligonucleotide primers in the context of double-stranded primer-template complex, nick-translation synthesis during oligonucleotide-directed mutagenesis and the degradation of the RNA component of RNA:DNA hybrids. When utilized in a PCR process with double-stranded primer-template complex, the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of a DNA polymerase may result in degradation of oligonucleotide primers from their 5xe2x80x2 end. This activity is undesirable not only in PCR, but also in second-strand cDNA synthesis and sequencing processes.
When choosing to produce and use an enzyme for sequencing, various factors are considered. For example, large quantities of the enzyme should be easy to prepare; the enzyme should be stable upon storage for considerable time periods; the enzyme should accept all deoxy and dideoxy nucleotides and analogues as substrates with equal affinities and high fidelity; the polymerase activity should be highly processive over nucleotide extensions to 1 kb and beyond, even through regions of secondary structure within the template; the activity should remain high, even in suboptimal conditions; and the enzyme should be inexpensive (A. T. Bankier, xe2x80x9cDideoxy sequencing reactions using Klenow fragment DNA polymerase I,xe2x80x9d in H. and A. Griffin (eds.), Methods in Molecular Biology: DNA Sequencing Protocols, Humana Press, Totowa, N.J., 1993, pp. 83-90). Furthermore, the enzyme should be able to function at elevated temperatures (e.g., greater than about 70xc2x0 C.), so that non-specific priming reactions are minimized. However, there are no native enzymes which fully meet all of these criteria. Thus, mutant forms of enzymes have been produced in order to address some of these needs.
For example, mutant forms of thermostable DNA polymerases that exhibit reduced or absent 5xe2x80x2 to 3xe2x80x2 exonuclease activity have been generated. The Stoffel fragment of Taq DNA polymerase lacks 5xe2x80x2 to 3xe2x80x2 exonuclease activity due to genetic manipulations that resulted in the production of a truncated protein lacking the N-terminal 289 amino acids (e.g., Lawyer et al., J. Biol. Chem., 264:6427-6437, 1989; and Lawyer et al., PCR Meth. Appl., 2:275-287, 1993). Analogous mutant polymerases have been generated from various polymerases, including Tma, Tsps17, TZ05, Tth and Taf. While the generation of thermostable polymerases lacking 5xe2x80x2 to 3xe2x80x2 exonuclease activity provides improved enzymes for certain applications, some of these mutant polymerases still have undesirable characteristics, including the presence of 3xe2x80x2 to 5xe2x80x2 exonuclease activity.
The 3xe2x80x2 to 5xe2x80x2 exonuclease activity is commonly referred to as proof-reading activity, it removes bases that are mismatched at the 3xe2x80x2 end of a primer in a primer-template duplex. While the presence of 3xe2x80x2 to 5xe2x80x2 exonuclease activity may be advantageous, as it leads to an increase in the fidelity of replication of nucleic acid strands, it also has some undesirable characteristics. The 3xe2x80x2 to 5xe2x80x2 exonuclease activity found in thermostable DNA polymerases such as Tma (including mutant forms of Tma that lack 5xe2x80x2 to 3xe2x80x2 exonuclease activity) also degrades single-stranded DNA such as primers used in PCR, single-stranded templates and single-stranded PCR products. The integrity of the 3xe2x80x2 end of an oligonucleotide primer used in a primer extension process (e.g., PCR, Sanger sequencing methods, etc.) is critical, as it is from this terminus that extension of the nascent strand begins. Degradation of the 3xe2x80x2 end of a primer results in loss of specificity in the priming reaction (i.e., the shorter the primer, the more likely that non-specific priming will occur).
Degradation of an oligonucleotide primer by a 3xe2x80x2 to 5xe2x80x2 exonuclease can be prevented by use of nucleotides modified at their 3xe2x80x2 terminus. For example, use of dideoxynucleotides or deoxynucleotides having a phosphorothiolate linkage between nucleotides at the 3xe2x80x2 terminus of an oligonucleotide can prevent degradation by 3xe2x80x2 to 5xe2x80x2 exonucleases. However, the need to use modified nucleotides to prevent degradation of oligonucleotides by a 3xe2x80x2 to 5xe2x80x2 exonuclease increases the time and cost required to prepare oligonucleotide primers.
A few examples of thermostable polymerases lacking both 5xe2x80x2 to 3xe2x80x2 exonuclease and 3xe2x80x2 to 5xe2x80x2 exonuclease are known. As discussed above, the Stoffel fragment of Taq DNA polymerase lacks the 5xe2x80x2 to 3xe2x80x2 exonuclease activity due to genetic manipulation and no 3xe2x80x2 to 5xe2x80x2 activity. is present, as Taq polymerase is naturally lacking in 3xe2x80x2 to 5xe2x80x2 exonuclease activity. Likewise, Tth polymerase naturally lacks 3xe2x80x2 to 5xe2x80x2 exonuclease activity and deletion nucleotide sequence encoding N-terminal amino acids can be used to remove 5xe2x80x2 to 3xe2x80x2 exonuclease activity.
Despite development of recombinant enzymes such as Stoffel fragment, there remains a need for other thermostable polymerases having improved characteristics for various applications. For example, some thermostable polymerases possess reverse transcriptase activity and they find use in reverse transcription methods since elevated temperatures help the enzyme to proceed through regions of the RNA which at lower temperatures would possess secondary structure. However, reverse transcription by thermostable DNA polymerases is often dependent on manganese. Unfortunately, the presence of manganese ions can cause higher rates of infidelity and damage to polynucleotides. Accordingly, what is needed in the art are improved thermostable DNA polymerases with enhanced properties, such as reverse transcriptase activity in the presence of magnesium.
The present invention relates to purified thermostable Thermoactinomyces vulgaris (Tvu) DNA polymerase. The present invention is not limited to any particular nucleic acid or amino acid sequence. Indeed, a variety of nucleic acid sequences encoding full-length, mutant, and truncated Tvu DNA polymerases are contemplated. The present invention also provides methods for the isolation of purified preparations of Tvu DNA polymerases. The origin of the Tvu DNA polymerases of the present invention is not limited to any particular source. Tvu DNA polymerases may be isolated from Tvu cells (i.e., native) or from host cells expressing nucleic acid sequences encoding Tvu DNA polymerase (i.e., recombinant).
In one embodiment, the present invention contemplates an isolated and purified, native thermostable Tvu DNA polymerase that has DNA synthesis activity. In another embodiment, the purified, native Tvu DNA polymerase has 5xe2x80x2 to 3xe2x80x2 exonuclease activity.
A contemplated isolated and purified, native Tvu DNA polymerase enzyme is at least 85 percent pure, in a more preferred embodiment the enzyme is at least 90 percent pure, and in a most preferred embodiment the enzyme is at least 95 percent pure, as determined by gel electrophoresis followed by staining or autoradiography then and laser scanning densitometry.
In another embodiment, the purified, native Tvu DNA polymerase exhibits reverse transcriptase activity in the presence of either magnesium ions or manganese ions. In a preferred embodiment, the purified, native Tvu DNA polymerase exhibits elevated reverse transcriptase activity in the presence of magnesium ions in comparison to reverse transcriptase activity in the presence of manganese ions. In still another embodiment, reverse transcriptase activity in the presence magnesium ions is manganese ion-independent.
In one embodiment, the present invention contemplates a purified, recombinant thermostable Tvu DNA polymerase that has DNA synthesis activity. In another embodiment, the purified, recombinant Tvu DNA polymerase has 5xe2x80x2 to 3xe2x80x2 exonuclease activity. A contemplated recombinant Tvu DNA polymerase has similar 5xe2x80x2 to 3xe2x80x2 exonuclease activity as compared to native Tvu DNA polymerase. In another embodiment, the recombinant Tvu DNA polymerase is mutant and has reduced 5xe2x80x2 to 3xe2x80x2 exonuclease activity as compared to the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of wild-type Tvu DNA polymerase. In another embodiment, the mutant Tvu polymerase is substantially free of 5xe2x80x2 to 3xe2x80x2 exonuclease activity.
In a preferred embodiment, the purified, recombinant Tvu DNA polymerase enzyme is at least 80 percent pure, in a more preferred embodiment, the enzyme is at least 90 percent pure, and in a most preferred embodiment, the enzyme is at least 95 percent pure, as determined by gel electrophoresis followed by staining or autoradiography and then laser scanning densitometry.
In another embodiment, the purified, recombinant Tvu DNA polymerase exhibits reverse transcriptase activity in the presence of either magnesium ions or manganese ions. In still other embodiments, reverse transcriptase activity in the presence magnesium ions is substantially manganese ion-independent.
The present invention further provides nucleic acids encoding thermostable Tvu DNA polymerases. The present invention is not limited to any particular form of nucleic acid. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA. Preferred contemplated Tvu DNA polymerase enzymes are encoded by the oligonucleotide having the sequence of SEQ ID NO: 1, or the truncated DNA coding sequence of SEQ ID NO: 3 or the truncated DNA coding sequence of SEQ ID NO: 5 or variants thereof.
However, the present invention is not limited to any one sequence. Indeed, a variety of variant nucleic acid sequences are contemplated. In some embodiments, the nucleic acid encoding thermostable Tvu DNA polymerases is mutated to encode a polymerase that is substantially free of 5xe2x80x2 to 3xe2x80x2 exonuclease activity. A DNA variant encoding Tvu DNA polymerase with DNA synthesis activity can have either conservative or non-conservative amino acid substitutions.
In some embodiments, the nucleic acid sequence is selected from sequences that hybridize to SEQ ID NO: 1 under high stringency conditions and sequences that hybridize to the complementary sequence of SEQ ID NO: 1 under high stringency conditions.
In other embodiments, the present invention provides purified oligonucleotides of at least 15 consecutive nucleotides of the nucleic acid of SEQ ID NO: 1 or complementary to at least 15 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO: 1.
In some embodiments, these oligonucleotides of at least 15 consecutive nucleotides of SEQ ID NO: 1 or its complement are used to amplify the nucleic acid of SEQ ID NO: 1 and variants or homologs thereof. In still other embodiments, the oligonucleotides are used to identify homologs or variants of the nucleic acid sequence of SEQ ID NO: 1 by hybridization procedures.
The present invention also provides recombinant DNA vectors or expression vectors comprising nucleic acid sequences that encode a thermostable Tvu DNA polymerase having DNA synthesis activity. In some embodiments, the polymerase-encoding nucleic acid sequence is set forth in SEQ ID NO: 1 or a DNA variant thereof. The DNA variant is as discussed above. In other embodiments, the recombinant DNA vector contains a mutant nucleic acid sequence set forth in SEQ ID NO: 3 and 5, or a DNA variant thereof, encoding a thermostable Tvu DNA polymerase that is substantially free of 5xe2x80x2 to 3xe2x80x2 exonuclease activity. A variant nucleic acid sequence is a sequence that encodes an amino acid residue sequence that is at least 95 percent or more identical to the sequence of a Tvu DNA polymerase of SEQ ID NOs. 2, 4, or 6.
In further embodiments, the vector comprises a recombinant nucleic acid selected from nucleic acids that hybridize to SEQ ID NO: 1, 3, or 5 or DNA variants thereof under conditions of medium or high stringency. In still further embodiments, the vector comprises a prokaryotic origin of replication. In other embodiments, the vector further comprises a promoter or enhancer sequence operably linked to the recombinant nucleic acid encoding Tvu DNA polymerase. Optionally, the promoter is inducible by an exogenously supplied agent, most preferably the promoter is induced by exogenously supplied IPTG. In some embodiments, the vector further comprises a selectable marker.
The present invention further contemplates host cells transformed with a vector comprising a nucleic acid sequence (or a variant thereof) encoding a Tvu DNA polymerase capable of DNA synthesis activity. The invention is not limited by the choice of host cell; host cells may comprise prokaryotic or eukaryotic cells. In some embodiments, the host cell is a bacterial cell (e.g., an E. Coli cell). In other embodiments the host cell is a mammalian cell, yeast cell, or insect cell.
The invention further provides methods for determining the DNA sequence of a segment or portion of a DNA molecule using the Tvu DNA polymerases of the invention. Traditional (i.e., Sanger) as well as other methods, including but not limited to, chain termination sequencing or thermal cycle sequencing protocols benefit from the use of the Tvu DNA polymerases of the invention. Thus, for example, in some embodiments, dideoxynucleotide (ddNTP) chain termination sequencing protocols are used in conjunction with the polymerases of the invention.
Accordingly, in some embodiments, the present invention provides methods for determining the nucleotide base sequence of a DNA molecule comprising the steps of a) providing in any order: i) a reaction vessel (e.g., any suitable container such as a microcentrifuge tube or a microtiter plate); ii) at least one deoxynucleoside triphosphate; iii) a thermostable Tvu DNA polymerase; iv) at least one DNA synthesis terminating agent that terminates DNA synthesis at a specific nucleotide base; v) a first DNA molecule; and vi) at least one primer capable of hybridizing to the first DNA molecule; b) adding to the reaction vessel, in any order, the deoxynucleoside triphosphate, DNA polymerase, DNA synthesis terminating agent, first DNA molecule, and the primer so as to form a reaction mixture, under conditions such that the primer hybridizes to the DNA molecule, and the DNA polymerase is capable of conducting primer extension to produce a population of DNA molecules complementary to the first DNA molecule; and c) determining at least a part of the nucleotide base sequence of the first DNA molecule. As the present invention encompasses any order of addition that permits the primer to hybridize to the DNA molecule and the DNA polymerase to be capable of conducting primer extension, the methods of the present invention are not limited by the order in which the reaction components are added to the reaction vessel. In a preferred embodiment, the DNA polymerase is added last. The conditions that permit the primer to hybridize to the DNA molecule, and allow the DNA polymerase to conduct primer extension may comprise the use of a buffer.
In one embodiment, the sequencing method uses a native Tvu DNA polymerase. In an alternative embodiment the sequencing method uses a recombinant DNA polymerase.
In an alternative embodiment, the conditions of the method comprise heating the mixture. In another embodiment, the method further comprises cooling the mixture to a temperature at which the thermostable DNA polymerase conducts primer extension. In a particularly preferred embodiment, the method further comprises one or more cycles of heating and then cooling. In yet another embodiment of the method, the reaction mixture comprises 7-deaza dGTP, dATP, dTTP and dCTP.
It is contemplated that various DNA synthesis terminating agents are useful in the present invention. In a preferred embodiment, the DNA synthesis terminating agent is a dideoxynucleoside triphosphate. In a particularly preferred embodiment, the dideoxynucleoside triphosphate is selected from the group consisting of ddGTP, ddATP, ddTTP and ddCTP.
It is also contemplated that the primer used in the sequencing method of the present invention is labelled. In a preferred embodiment, the primer is labelled with 32P, 33P, 35S, enzyme, or fluorescent molecule. It is also contemplated that reactants other than the primer used in the method of the present invention are labelled. For example, in one embodiment, one deoxynucleoside triphosphate is labelled. In a preferred form of this embodiment, the deoxynucleoside triphosphate is labelled with 32P, 33P, 35S, enzyme, or a fluorescent molecule.
It is further contemplated that additional steps or sub-steps will be incorporated into the sequencing method of the present invention. For example, in one embodiment, step b) further comprises adding a thermostable pyrophosphatase to the reaction mixture. In a preferred form of this embodiment, the thermostable pyrophosphatase is Thermus thermophilus pyrophosphatase. In some embodiments, the method uses a mixture or blend comprising a Tvu DNA polymerase and a thermostable pyrophosphatase.
The present invention also provides kits, for example, for determining the nucleotide base sequence of a DNA molecule comprising: a) a thermostable Tvu DNA polymerase; and b) at least one nucleotide mixture comprising deoxynucleoside triphosphates and one dideoxynucleoside triphosphate. In a preferred embodiment, the polymerase of the kit is a non-naturally occurring DNA polymerase. It is also contemplated that the mutant Tvu DNA polymerase is substantially free of significant 5xe2x80x2 exonuclease activity. In another embodiment, the mutant Tvu DNA polymerase of the kit is substantially free of 3xe2x80x2 exonuclease activity.
In an alternative embodiment, the kit of the present invention contains a first nucleotide mixture, a second nucleotide mixture, a third nucleotide mixture, and a fourth nucleotide mixture, with the first nucleotide mixture comprising ddGTP, 7-deaza dGTP, dATP, dTTP and dCTP, the second nucleotide mixture comprising ddATP, 7-deaza dGTP, dATP, dTTP and dCTP, the third nucleotide mixture comprising ddTTP, 7-deaza dGTP, dATP, dTTP and dCTP and the fourth nucleotide mixture ddCTP, 7-deaza dGTP, dATP, dTTP and dCTP. It is also contemplated that the kit of this embodiment further comprises a thermostable pyrophosphatase. In a particularly preferred embodiment, the thermostable pyrophosphatase is Tth pyrophosphatase. In preferred embodiments, the kit contains a mixture or blend comprising a Tvu DNA polymerase and a thermostable pyrophosphatase.
The present invention also provides methods for amplifying a double stranded DNA molecule, comprising the steps of: a) providing: i) a first DNA molecule comprising a first strand and a second strand, wherein the first and second strands are complementary to one another; ii) a first primer and a second primer, wherein the first primer is complementary to the first DNA strand, and the second primer is complementary to the second DNA strand; and iii) a first thermostable DNA polymerase derived from the eubacterium Thermoactinomyces vulgaris; and b) mixing the first DNA molecule, first primer, second primer, and polymerase to form a reaction mixture under conditions such that a second DNA molecule comprising a third strand and a fourth strand are synthesized, with the third strand having a region complementary to the first strand and the fourth strand having a region complementary to the second strand. The method of the present invention is not limited by the source of the first DNA molecule. In a preferred embodiment, the first DNA molecule is present in a genomic DNA mixture (e.g., in genomic DNA extracted from an organism, tissue or cell line). In alternative embodiments, the first DNA molecule is derived from an RNA molecule by means of reverse transcription (RT). The newly synthesized DNA molecule (cDNA) then serves as substrate in a subsequent amplification reaction (PCR). The conditions that permit the primer to hybridize to the DNA molecule, and allow the DNA polymerase, either alone or in combination with another thermostable DNA polymerase, to conduct primer extension may comprise the use of a buffer.
In one embodiment, the method conditions comprise heating the mixture. In an alternative embodiment, the method further comprises cooling the mixture to a temperature at which the thermostable Tvu DNA polymerase, either alone or in combination with another thermostable DNA polymerase, can conduct primer extension. In a particularly preferred embodiment, the method comprises repeating the heating and cooling the mixture one or more times.
It is also contemplated that the Tvu DNA polymerase of the method will have various properties. It is therefore contemplated that in one embodiment of the method, the polymerase is substantially free of 5xe2x80x2 to 3xe2x80x2 exonuclease activity. In another embodiment, the polymerase is substantially free of both 5xe2x80x2 to 3xe2x80x2 exonuclease and 3 to 5xe2x80x2 exonuclease activity. In other embodiments, the polymerase has reverse transcriptase activity in the presence of either magnesium or manganese ions. In still other embodiments, the reverse transcriptase activity in presence of magnesium ions is substantially manganese ion-independent.
The present invention has many benefits and advantages, several of which are listed below.
One benefit of the invention is that the thermostable Tvu DNA polymerase enzyme can be used for processes of high temperature nucleic acid amplification and sequencing without substantial loss of DNA synthesis activity.
An advantage of the invention is that the enzyme can be used to perform high temperature reverse transcription in the absence of manganese ions.
A further advantage of the invention is that the enzyme can be used in high throughput robotically-manipulated procedures because greater enzymatic stability is retained at room temperature.
Still further benefits and advantages will be apparent to the worker of ordinary skill from the disclosure that follows.